EVALUATION OF FERN AND MOSS PROTEIN-BASED DEFENSES AGAINST PHYTOPHAGOUS INSECTS

Int. J. Plant Sci. 167(1):111–117. 2006. Ó 2006 by The University of Chicago. All rights reserved. 1058-5893/2006/16701-0010$15.00 EVALUATION OF FERN...
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Int. J. Plant Sci. 167(1):111–117. 2006. Ó 2006 by The University of Chicago. All rights reserved. 1058-5893/2006/16701-0010$15.00

EVALUATION OF FERN AND MOSS PROTEIN-BASED DEFENSES AGAINST PHYTOPHAGOUS INSECTS Kevin Markham,* Tanya Chalk,* and C. Neal Stewart Jr.1 ,y *University of North Carolina, Greensboro, North Carolina 27403, U.S.A.; and yUniversity of Tennessee, Department of Plant Sciences, Knoxville, Tennessee 37996, U.S.A.

Relatively little is known about insect defense mechanisms in the ferns and mosses. The current paradigm is that secondary metabolites and physical barriers are most important in conferring insect resistance in ferns, lycopods, and mosses. We investigated whether protein-based resistance exists in representatives of these taxa. We screened a total of 23 plant species for protein-based insecticidal activity against the two common lepidopteran pests: corn earworm (Helicoverpa zea) and fall armyworm (Spodoptera frugiperda). Protein extracts from fern and moss species were compared with those from a lepidopteran-susceptible soybean (Glycine max) cultivar (Cobb) in bioassays for insect resistance. The ebony spleenwort (Asplenium platyneuron), sensitive fern (Onoclea sensibilis), glade fern (Anthyrium pycnocarpon), and the burned ground moss (Ceratodon purpureus) protein extracts caused the greatest decrease in damage in leaf-disk assays and insect larval growth. These species are good candidates for follow-up evaluation. Keywords: insect resistance proteins, ferns, mosses, lepidoptera.

Introduction

vore that utilizes them as food sources. Lepidopterans that eat ferns or mosses are especially rare (Weintraub 1995), yet caterpillars, which possess chewing mouthparts, cause extensive damage to crops. Those insects that do utilize lower plants as a food source often possess piercing-sucking mouthparts that enable them to bypass the high concentration of secondary metabolites found in lower plant cell walls by inserting the mouthparts directly into phloem and sucking sap. Auerbach and Hendrix (1980) suggest that the observation of ferns being underutilized by insects in comparison to angiosperms might result from the lack of flowers or fruit in ferns. Hendrix and Marquis (1983) also found that the vegetative portions of ferns are fed upon by insects to the same extent as angiosperm vegetative tissue. Ferns, fern allies, and mosses all produce many secondary metabolites (Asakawa 1990). It has been the general assumption that these secondary compounds are the primary mechanism of lower plants’ insect resistance (IR). Ferulic acid, hydrolysable tannins, terpenes, and alkaloids have been isolated from lower plants (Schaufelberger and Hostettmann 1983; Asakawa 1990). These same secondary metabolites have been shown to deter insect pests in higher plants and would be expected to demonstrate the same properties in lower plants (Rosenthal 1982; Turlings and Tumlinson 1992; Basra and Basra 1997). Other secondary metabolites such as pterosins, phenolic acids, sulfated cinnamic acids, and flavonoids isolated from various lower plants provide effective resistance to feeding insects (McMorris et al. 1977; Davidson et al. 1989; Enyedi et al. 1992; Harborne 1993). Proanthocyanidins are believed to be the most effective broad-spectrum defense against fern predators (CooperDriver 1985). Ecdysones have also been isolated from ferns and are believed to deter feeding by mimicking insect molting hormones (Jones and Firn 1978; Lafont and Horn 1989).

Ferns, lycopods, and mosses are members of a large and diverse group of plants commonly referred to as the lower plants. Lower plants are comparable to the angiosperms in their species diversity, yet relatively little is known about their physiology and genetics. In fact, it has been estimated that only 5% of all bryophytes have been studied with regard to any phytochemical properties (Asakawa 2001). Even less is known about the genomics, proteomics, and biochemical pathways of ferns and mosses. Although these taxa show rich promise in unlocking physiological properties such as drought tolerance, insect resistance, disease resistance, and tolerance to heavy metals, there is still little gene discovery research being performed on ferns and mosses. This may be because these plants are of little economic importance to commercial agriculture. However, one notable exception is Physcomitrella patens, which has been the subject of concerted genomic research as the model moss (Rensing et al. 2002; Nishiyama et al. 2003). Although 9300 insect species have been estimated to use ferns as a food source (Cooper-Driver 1978), there is a predominant school of thought that ferns and mosses are rarely fed on by phytophagous insects in nature (Eastop 1973; Swain and Cooper-Driver 1973; Hendrix 1980; Davidson et al. 1989). Angiosperms, in comparison, are used as food for ca. 400,000 species of insects. Hendrix (1980) estimated that the ratio of insect herbivore species to angiosperm species is approximately 1 : 1 (although it is probably closer to 2 : 1, given current estimates of angiosperm extant species). In contrast, for every 24 ferns there is only one insect herbi1

Author for correspondence; e-mail [email protected].

Manuscript received April 2005; revised manuscript received August 2005.

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A literature search yielded few articles on the subject of fern and moss IR proteins. Apparently, thiaminase has been the only fern or moss plant enzyme shown to demonstrate IR activity. Thiaminase is a fern enzyme associated with and the major causative agent of bracken poisoning and B1 deficiency in cattle and other ruminants (Fenwick 1988). Hendrix (1977) showed that thiaminase deterred feeding by the southern armyworm in the fern Nephrolepis exaltata. Our study is an attempt at describing broad protein-based IR in ferns and mosses. Twenty-three species were screened for protein-based IR against two common lepidopteran crop pests: Helicoverpa zea and Spodoptera frugiperda. These insects were chosen because of their economic importance and commercial availability. Protein-based defenses against pest insects on higher plants have been well documented (reviewed in Basra and Basra 1997). It would be expected that lower plants might have similar capabilities. Because mosses are evolutionarily older than higher plants, it would also be interesting to deduce the evolutionary origins of protein-based insect defenses in land plants. Any findings in this field will increase our knowledge of plant/insect coevolution and add to our understanding of lower plant insect defenses.

Material and Methods Plant Material Seventeen fern and six moss species commonly growing wild in North Carolina were used for a functional screen (table 1). Soybean (Glycine max cv. Cobb), which is a suitable food source for lepidopteran larvae, was used as a negative control. One gram of newly emerged and fully expanded (in April–May) fronds was sampled from plants growing naturally in various field locations in North Carolina, and the tissue was placed into a sterile, prechilled 1.5-mL centrifuge tube and placed immediately on ice for transport back to the laboratory, which took less than 1 h. Moss tissue of unknown age, also collected in April–May, included both the sporophyte and gametophyte portions of the plant (i.e., whole plants). Moss tissue was collected at least 48 h after the last rain, which we found allowed for easier homogenization of tissue. A total of 15 g of tissue was collected for each plant species.

Total Protein Extraction In the laboratory, samples were flash frozen in liquid nitrogen for 1 min and immediately stored at 80°C until homogenization. Tissue was crushed into a fine powder and then placed directly on ice. Powdered tissue was resuspended in 1 mL of cold (4°C) protein-extraction buffer (20 mM Hepes pH 8, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine). The homogenate was incubated on ice for 1 h with occasional mixing. The samples were then centrifuged at 3000 g at 4°C for 30 min. The supernatant was collected into a cold sterile 1.5-mL centrifuge tube using cold sterile 1-mL pipette tips. Protein extracts were concentrated by ammonium sulfate (55%) precipitation and were centrifuged at 3000 g for 30 min, and the pellet was resuspended in 0.5 mL of extraction buffer. Extracts for each plant tissue were combined (7.5 mL) into a cold 15-mL centrifuge tube and dialyzed using a cellu-

Table 1 Summary of the 23 Fern and Moss Species Sampled and Their Corresponding Protein Yields in Micrograms as Determined by Spectrophotometric Analysis at A280

Species Ferns: Cinnamon fern (Osmunda cinnamomea L.) Rattlesnake fern (Botrychium virginianum (L.) Swartz)* American climbing fern (Lygodium palmatum (Bernh.) Swartz) Resurrection fern (Polypodium polypodioides (L.) Watt)* Bracken fern (Pteridium aquilinum (L.) Kuhn)* Broad beech fern (Thelypteris hexagonoptera (Michx.) Weath.) Ebony spleenwort (Asplenium platyneuron (L.) BSP.)* Lady fern (Athyrium filix L.) Fragile fern (Cystopteris fragilis (L.) Bernh.)* Christmas fern (Polystichum acrostichoides (Michx.) Schott)* Sensitive fern (Onoclea sensibilis L.)* Netted chain fern (Woodwardia areolata (L.) Moore)* Oak fern (Gymnocarpium dryopteris (L.) Newmn.)* Scouring rush (Equisetum hyemale L.) Royal fern (Osmunda regalis L.) Glade fern (Athyrium pycnocarpon (Spreng.) Tidstr.)* Mosquito fern (Azolla caroliniana Willd.) Lycopods: Shining club moss (Lycopodium lucidulum Michx.) Meadow spike moss (Selaginella apoda L.) Mosses: Burned ground moss (Ceratodon purpureus (Hedw.) Brid)* Water fern moss (Fissiden grandifrons Brid.) Red spoonleaf peat moss (Sphagnum magellanicum Brid.) Silver moss (Bryum argenteum L.)

Protein concentration Total protein (mg/mL) yield (mg)

0.15

300

4.2

8400

0

0

2.55

5100

3.72

7440

0.15

300

4.21 0

8420 0

3.54

7080

2.35 4.25

4700 8500

2.75

5500

5.1 0.55 0

10200 1100 0

2.75

5500

0.15

300

0.23

460

0

0

3.05

6100

0.13

250

0 0

0 0

Note. Asterisks denote 11 samples that yielded more than 2 mg/mL of protein, the amount required for bioassay experiments.

lose membrane with an exclusion limit of

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